ABSTRACT
The phenanthridine alkaloid lycobetaine is a minor constituent of Amaryllidaceae. Inhibition of cell growth was studied in the clonogenic assay on 21 human tumour xenografts (mean IC(50) = 0.8 microM). The growth of human leukaemia cell lines was also potently inhibited (mean IC(50) = 1.3 microM). Athymic nude mice, carrying s.c. implanted human gastric tumour xenograft GXF251, were treated i.p. with lycobetaine for 4 weeks, resulting in a marked tumour growth delay. Lycobetaine was found to act as a specific topoisomerase II beta poison. In the presence of calf thymus DNA, pure recombinant human topoisomerase II beta protein was selectively depleted from SDS-gels, whereas no depletion of topoisomerase II alpha protein was observed. In A431 cells immunoband-depletion of topoisomerase II beta was induced, suggesting stabilization of the covalent catalytic DNA-intermediate in living cells. It is reasonable to assume that this mechanism will cause or at least contribute significantly to the antitumour activity.
Subject(s)
Alkaloids/pharmacology , Amaryllidaceae Alkaloids , Antineoplastic Agents, Phytogenic/pharmacology , Enzyme Inhibitors/pharmacology , Indolizines , Neoplasms, Experimental/drug therapy , Topoisomerase II Inhibitors , Alkaloids/chemistry , Animals , Antigens, Neoplasm , Antineoplastic Agents, Phytogenic/chemistry , Blotting, Western , Cell Cycle , Cell Division/drug effects , Comet Assay , DNA/metabolism , DNA Damage , DNA Topoisomerases, Type II/immunology , DNA Topoisomerases, Type II/metabolism , DNA-Binding Proteins , Enzyme Inhibitors/chemistry , HL-60 Cells , Humans , Mice , Mice, Nude , Neoplasms, Experimental/enzymology , Neoplasms, Experimental/pathology , Tumor Cells, Cultured , Tumor Stem Cell Assay , Xenograft Model Antitumor AssaysABSTRACT
Recently, we reported that the monoclonal antibody specific for human DNA topoisomerase IIalpha, Ki-S1, stains not only the nuclei of human A431 cells but also extranuclear structures suggestive of centrosomes (Meyer, K. N., Kjeldsen, E., Straub, T., Knudsen, B. K., Kikuchi, A., Hickson, I. D., Kreipe, H., and Boege, F. (1997) J. Cell Biol. 136, 775-788). Here, we confirm colocalization of Ki-S1 with the centrosomal marker gamma-tubulin. In addition, we show labeling of centrosomes by peptide antibodies against the N and C termini of human topoisomerase IIalpha. Probing Western blots of isolated centrosomes with topoisomerase IIalpha antibodies, we demonstrate a protein band of 170 kDa. Moreover, isolated centrosomes exhibited DNA decatenation and relaxation activity correlated to the amount of topoisomerase IIalpha protein in the same way as seen in the pure recombinant enzyme. Topoisomerase IIalpha epitopes could not be removed from centrosomes by salt extraction, DNase treatment, or RNase treatment, procedures that completely removed the enzyme from nuclei. Taken together, these observations suggest that active topoisomerase IIalpha is bound tightly to the centrosome in a DNA-independent manner. Because such centrosomal topoisomerase IIalpha was also present in quiescent lymphocytes devoid of topoisomerase IIalpha in the nuclei, we assume that it might be a long-lived storage form.
